Method for increasing the concentration of conjugated linoleic acid in milk and/or tissue fat of a ruminant

ABSTRACT

The invention relates to a method for increasing the concentration of the cis-9, trans-11 isomer of octadecadienoic acid in the milk fat and/or the tissue fat of a ruminant. In the method the ruminant is fed the trans-11 isomer of octadecenoic acid either as such or mixed with other feed, separately or together with other fatty acids.

The invention relates to a method for increasing the concentration ofconjugated linoleic acid, i.e. chemically precisely expressed thecis-9,trans-11 isomer of octadecadienoic acid (CLA,cis-9,trans-11-C18:2) in the milk fat and/or the tissue fat of aruminant by feeding to the ruminant, either as such or mixed with otherfeed, separately or together with other fatty acids, vaccenic acid, i.e.chemically precisely expressed the trans-11 isomer of octadecenoic acid(trans-11-C18:1).

Recently it has been observed that conjugated linoleic acid, i.e. CLA,provides quite effective protection against several forms of cancer (HaY. L. et al., Cancer Res. 1990, 50:1097; Ip C. et al., Cancer 1994,74:1050). It has been observed to be effective against skin and stomachcancers of the mouse and against cancer of the mammary gland of the rat(Scimeca J. A. et al., Diet and Breast Cancer. American Inst. Of CancerRes. 1994, Plenum Press, London). The growth of human cancer cells hasalso successfully been inhibited in cell cultures (Shultz T. D. et al.,Canc. lett. 1992, 63:125). It has also been observed to have othermetabolic effects, some of which clearly suggest effects on health(Banni, S. and J.-C. Martin. 1998. Trans fatty acids in human nutrition.Ed. Sebedio and Christie, The Oily Press, Dundee).

In nature, CLA is present mainly as a component in the tissue fat andthe milk fat of ruminants, cis-9,trans-11 being the main isomer (approx.90%). Small amounts of other isomers are also present in milk, but inthe present context, CLA denotes specifically the cis-9,trans-11 isomer.

It is thus an object of the present invention to provide a methodsuitable for animal husbandry by which the concentration of CLA in themilk fat and/or the tissue fat of a ruminant can be increased. This hasbeen achieved according to the invention in the manner disclosed in theaccompanying patent claims.

The method according to the present invention is suitable for allruminants, in particular for lactating ruminants, such as the cow,whereby it is possible to produce for consumers milk and milk productswith a modified fatty acid composition. The change in the fatty acidcomposition of milk and of milk products is advantageous for the healthof consumers. Usually CLA, i.e. cis-9,trans-11-C18:2 fatty acid,constitutes 0.3-0.7% by weight of all of the fatty acids in cow's milk.Even at this concentration it may already protect people consuming milkproducts from cancer, if the intake of milk fat is sufficiently high(Knekt P. R. et al., Brit. J. of Cancer 1996, 73:687). Habits in theconsumption of food fats have changed in recent years. The consumptionof fats, and in particular milk fat, has decreased, and as a consequenceof this also the intake of CLA in food has decreased. By increasing CLAconcentration in milk by amount corresponding to the decrease in theconsumption of milk fat it would be possible to return the intake of CLAin food to the level of previous years without needing to increase theconsumption of fats. The increasing of the concentration of CLA in milkmay thus have a great significance for public health. Increasing theconcentration of CLA in milk to a level higher than previously couldhave an especially positive effect, since in experiments on animals ithas been observed that the protective effect of CLA has a clear doseresponse (Ip C. et al., Cancer 1994, 74:1050). CLA in food may alsolower the amount of bad cholesterol (LDL) in the blood (Lee et al. 1994.Atherosclerosis. 108:19). The natural CLA isomer of milk fat has beeningested by people for thousands of years without any observeddisadvantages.

The formation of conjugated linoleic acid (cis-9,trans-11-C18:2) duringthe isomerization of linoleic acid (cis-9,cis-12-C18:2) in the rumen hasbeen described as follows:

The concentration of CLA in milk has successfully been increased byfeeding cows vegetable oils which contain linoleic acid (Griinari J. M.et al., J. Dairy Sci. 1998, 81:1251). Kelly et al. (Kelly M. L. et al.,J. Nutr. 1998, 128:881) compared three vegetable oil additions to feedand their effect on milk CLA concentration and observed that the oiladdition with the highest concentration of linoleic acid (sunflower oil)gave the best milk CLA response. They noted that the concentration ofCLA in milk can be increased by feeding to animals oily fatsspecifically containing linoleic acid.

It has been believed that the CLA which has passed into milk fat isderived solely from the linoleic acid present in animal feed, i.e. fromoctadecadienoic acid cis-9,cis-12 isomer, which the microbes of therumen isomerize to cis-9,trans-11-C18:2 before reducing it to vaccenicacid (trans-11-C18:1) and further to stearic acid (C18:0). CLA is notformed in notable amounts in the hydrogenation of linolenic acid, i.e.the cis-9,cis-12,cis-15-C18:3 isomer of octadecatrienoic acid, butvaccenic acid is also an important biohydrogenation product of linolenicacid (Harfoot and Hazlewood 1988, The rumen microbial ecosystem. Ed.Hobson, P. N. Elsevier Science Publishers B. V., Amsterdam, TheNetherlands). According to the prevailing theory, some of the CLA formedas an intermediate product in the biohydrogenation of linoleic acidescapes from the rumen along with the continuous outflow of the rumenfluid before it becomes reduced to vaccenic acid. The CLA which hasescaped from the rumen is absorbed mainly in the small intestine, istransferred by the blood circulation to the mammary gland and is boundthere as part of the fatty acid composition of milk fat.

The authors of the present invention observed that fish oil, whichcontains low levels of linoleic acid (composition shown in Table 1),increased the CLA concentration in milk (Table 2).

Table 1 Fatty acid composition of fish oil

Myristic acid (14:0) 8.6 Palmitic acid (16:0) 18.6 Palmitoleic acid(cis-9-16:1) 11.9 Stearic acid (18:0) 3.6 Oleic acid (cis-9-18:1) 9.4Other cis-acids 0.9 Cis-Vaccenic acid (cis-11-18:1) 5.2 Elaidic acid(trans-9-18:1) 0.2 Linoleic acid (18:2, n-6) 2.5 Linolenic acid (18:3,n-3) 1.7 EPA (20:5, n-3) 10.2 DHA (22:6, n-3) 7.6

TABLE 2 Effect of the feeding of fish oil on the fatty acid compositionof milk (% of total fatty acids) No oil Fish oil addition addition C12:03.4 1.9 C14:0 12.8 9.3 C16:0 38.1 34.3 C18:0 9.7 3.8 C18:1 26.1 28.6trans-11-C18:1 1.1 3.2 C18:2 2.4 2.6 C18:3 0.2 0.5 CLA 0.7 1.9

The generally approved mechanism of CLA formation described above(Harfoot and Hazlewood 1988) also does not explain the increase of theconcentration of CLA in the milk when cows receive pasture grass astheir only feed (Griinari et al. 1998 J. Dairy Sci. 81 (Suppl. 1):300(Abst.)), since the principal fatty acid in the fat present in pasturegrass is linolenic acid and the proportion of linoleic acid is typicallyquite low.

Based on observations cited above, the present inventors drew theconclusion that the CLA in milk may also be derived from a source otherthan the linoleic acid isomerization taking place in the rumen.Furthermore, since in the rumen of a cow feeding on nothing but pasturegrass certain CLA isomers (cis-9,trans-11-C18:2 andtrans-9,trans-11-C:18:2) were present at an average ratio of 1:1, but inmilk the concentration of the cis-9,trans-11-C18:2 isomer was 10 timeshigher than that of trans-9,trans-11-C:18:2 isomer (Griinari andNumeral, unpublished), the present inventors noted that the CLA of milkfat may in a certain feeding situation be formed to a large extentelsewhere than in the rumen. It has been observed that theabove-mentioned CLA isomers transfer from the digestive tract to themilk at almost the same rate, so that what is involved in this situationis not the discrimination of one of the fatty acid isomers (Chouinard etal. 1998. J. Dairy Sci. 81 (Suppl. 1):353 (Abst.)).

Thus the present invention is based on the surprising observation thatan oil addition to feed containing low levels of linoleic acid caused anotable increase in the concentration of CLA in the milk. The initialsubstance of CLA must, however, be formed in the rumen, since CLA ispresent in considerable amounts in the milk fat and the tissue fat ofruminants only.

It has also been observed in connection with the present invention thatin a cow feeding only on pasture grass the concentration of severaltrans-fatty acids (i.e. fatty acid isomers formed in the rumen) in thedigesta flowing out of the rumen closely predicts the concentrations ofthese fatty acids in milk (FIG. 1). The close correlation between theconcentration in the rumen digesta and the concentration in the milk fatsupports the hypothesis of the rumen origin of these fatty acid isomers.As regards CLA, the said correlation is, however, not true (FIG. 2a).The concentration of vaccenic acid (trans-11) in the digested feedflowing out of the rumen in fact explains the variation in theconcentration of CLA in the milk considerably better (FIG. 2b). Anexamination of the relationship of vaccenic acid to CLA in milk fatshows that the correlation is even closer (FIG. 2c). This very closecorrelation is probably explained as a metabolic substrate(trans-11-C18:1) and product (cis-9,trans-11-C18:2) relationship.

On the basis of the above examination of the correlation and the relatedconclusion, the surprising observation was made in connection with thepresent invention that the milk CLA may in certain feeding conditions beformed mainly through the desaturation of vaccenic acid to CLA. The saidconversion takes place under the action of the Δ-9 desaturase enzyme inthe tissues of the animal, as follows:

Δ-9 desaturase trans-11-C18:1 (vaccenic acid) cis-9,trans-11-C18:2 (CLA)

Thus it is possible that the CLA of milk fat is mainly formed in thetissues of the ruminant as Δ-9 desaturase adds a cis-9 double bond tothe trans-11-C18:1 fatty acid which forms in the rumen and escapes fromthe rumen along with digested feed. Δ-9 desaturase is an active enzymein a number of different tissues, but its activity has been observed inparticular in the cow's mammary gland, where it desaturates stearic acid(C18:0) to oleic acid (cis-9-C18:1) (Kinsella J. E., Lipids 1972,7:349). On the other hand the microsomal Δ-9 desaturase in the livercells of the rat has been observed to desaturate trans-11-C18:1 fattyacid to cis-9,trans-11-CLA in vitro (Mahfouz M. M. et al., Biochim.Biophys. Acta 1980, 618:1).

Feeding which increases the formation of vaccenic acid, i.e.trans-11-C18:1 fatty acid, in the rumen may thus, owing to thedesaturation of the trans-11-C18:1 fatty acid in the mammary gland orelsewhere in the cow's organism, increase the CLA concentration in themilk. The above-mentioned effect of fish oil increasing the CLAconcentration in milk would thus also be explainable by the effect offish oil on the rumen synthesis of trans-11-C18:1 fatty acid. This issupported by the observation of Pennington and Davis (J. Dairy Sci.1975. 58:49) on an increased rumen synthesis of vaccenic acid when fishoil was fed to cows. According to the present invention, the increasingof the CLA concentration in milk in certain feeding situations islargely explained by the formation of trans-11-C18:1 fatty acid in therumen and the desaturation of the said fatty acid to CLA in the organismof the cow, and thus not by the formation of CLA in the rumen as hasbeen believed so far (Harfoot and Hazlewood 1988). The previouslydescribed enrichment of the cis-9,trans-11-CLA isomer in milk inproportion to the concentration of the trans-9,trans-11-CLA isomer inpasture-fed cows is explained by the fact that the trans-9,trans-11isomer cannot be formed in cow tissues, whereas the cis-9,trans-11isomer could be mainly formed as a product of desaturation.Respectively, with pasture feeding perhaps only a small proportion ofthe cis-9,trans-11-CLA of the milk is formed in the rumen.

The present invention is based on the increasing of the concentration ofCLA in milk fat and tissue fat by feeding the ruminant a fat supplementwhich contains trans-11-C18:1 fatty acid, i.e. vaccenic acid, i.e. thesubstrate of the natural Δ-9 desaturase of the organism. Vaccenic acidis converted in the organism, for example in the mammary gland, tocis-9,trans-11-CLA and is secreted to the milk. Similarly, CLA formed inthe tissues can be incorporated to tissue lipids.

U.S. Pat. No. 5,416,115 discloses a method by which cow milk fats can bedecreased and milk production increased by the use of trans-fatty acids.In the method, for example, a trans-18:1 isomer blend was infused into acow. However, this publication did not state that the trans-18:1 fattyacid supplement in animal feed would increase the concentration of CLAin the milk fat and the tissue fat.

Food which contains trans-11-C18:1 fatty acid has been found to increasethe concentration of cis-9,trans-11 CLA in the blood (Salminen et al. J.Nutr. Biochem. 1998. 9:93) when the diet of the test subjects contained,in a manner deviating from the conventional diet, a very small quantityof polyunsaturated fatty acids. The increase in blood CLA concentrationreported by Salminen et al. (1998) shows that the trans-fatty aciddesaturation previously observed in liver cells (Mahfouz M. M. et al.,Biochim. Biophys. Acta 1980, 618:1) is an active process also in humansubjects, but limits it to diets which contain limited amounts ofpolyunsaturated fatty acids. Santora et al. (1998) (paper presented atthe annual meeting of AOCS, Chicago) noted that, when vaccenic acid wasfed to mice, their tissue fat CLA concentration tripled. The formationof CLA decreased when the feed of the mice contained 5% corn oil. Cornoil contains large amounts of polyunsaturated fatty acids, i.e. theactivity of desaturase is dependent on the degree of unsaturation of thediet.

The present invention shows that a ruminant can be used for theproduction of the naturally most common CLA isomer for human food byadding vaccenic acid (trans-11-C18:1) to the ruminant's feed. Theruminant is preferably a lactating ruminant, such as a cow, but theinvention can also be applied to ruminants intended for meat production.

In the feeding of a ruminant it is advantageous to protect, by knownprotection methods, the trans-11-C18:1 fat from the effects of rumenbiohydrogenation. For example, U.S. Pat. No. 4,642,317 discloses acommonly used method for protecting the fat.

In order to increase the CLA concentration in milk fat it isadvantageous to feed vaccenic acid, i.e. trans-11-C18:1 fatty acid, in amixture which contains large amounts of saturated fatty acids, since thepassing of saturated fatty acids to the mammary gland promotes theactivity of the mammary gland Δ-9 desaturase and may thereby alsopromote the conversion of the trans-11 isomer to cis-9,trans-11 CLA.

The most advantageous way of dosing vaccenic acid to animals is to feedit mixed with feed or as a separate feed supplement. The preferabledoses of vaccenic acid to be added can be determined by measuring avaccenic acid amount by which an optimal CLA level is obtained in milkso that the addition is sufficiently economical in terms of costs.

The invention is illustrated below with the help of examples.

EXAMPLE 1

The purpose of the experiment was to show that trans-11-C18: 1 dosed tolactating cows post ruminally into the digestive tract was converted tocis-9,trans-11-CLA in the tissues. The experiment was carried out byadministering fat-free milk (carrier, 5 kg/d) to the cows by continuousinfusion into the abomasum for three days (Preliminary Period). For thenext three days (Infusion) 25 g/d of a mixture of trans-11 fatty acidand trans-12-C18:1 fatty acid, emulsified in fat-free milk, was infusedinto the cows. The concentrations of trans-11-C18:1 and CLA weredetermined from the milk of the cows on the third day of both thePreliminary Sequence and of the Infusion. The results are shown inaccompanying Table 3.

TABLE 3 Preliminary % Period Infusion Increase Milk fatty acid (mg/g ofmilk fat) trans-11-C18:1 12.0 14.0 17 cis-9, trans-11-C18:2 2.8 4.1 46

The concentration of trans-11-C18:1 in the milk increased duringinfusion, showing that the infused fatty acid had been absorbed, hadpassed into the mammary gland and had become part of the milk fat. TheCLA concentration also increased, showing that a significant portion ofthe trans-11-C18:1 fatty acid received by the animal had been convertedin the cow's tissues to CLA. This experiment shows that ruminants, whichnaturally produce CLA in their body, increase the production of CLA ifthey receive trans-11-C18:1 fatty acid in an absorbable form, forexample added to their feed. Trans-11-C18:1 fatty acid must be dosedinto the feed daily, since the CLA concentration in the milk decreasesimmediately when the feeding is discontinued. In this experiment the CLAconcentration in the milk dropped to the basal level in two days afterthe infusion of the trans-fatty acids had been discontinued (FIG. 3).

EXAMPLE 2

Dairy cows (n=2) were fed a hardened vegetable fat mixture (0, 200 or400 g/d), which contained 7.9% trans-11-C18: 1 fatty acid. The CLAconcentration in the milk increased when the feeding of hardenedvegetable fats was increased (Table 4). This experiment demonstrates adose response effect between the dietary intake of trans-11-C18:1 fattyacid and the CLA concentration in the cow's milk.

TABLE 4 Control, 0 g/d 200 g/d 400 g/d In milk fatty acids, %trans-11-C18:1 1.2 2.1 2.6 cis-9, trans-11-C18:2 0.5 1.0 1.4

What is claimed is:
 1. A method for increasing the concentration of thecis-9,trans-11 isomer of octadecadienoic acid (CLA) in the milk fatand/or the tissue fat of a ruminant, characterized in that the trans-11isomer of octadecenoic acid is fed to the ruminant in an amount and fora period of time effective to increase the CLA level to above thatnormally present in the milk fat and/or tissue fat of a ruminant, eitheras such or mixed with other feed, separately or together with otherfatty acids.
 2. A method according to claim 1, characterized in that theruminant is a lactating ruminant.
 3. A method according to claim 1 or 2,characterized in that the trans-11 isomer of octadecenoic acid isprotected from reduction in the rumen.
 4. A method according to claim 1,characterized in that the other fatty acids are saturated fatty acids.5. A method for increasing the concentration of the cis-9,trans-11isomer of octadecadienoic acid (CLA) in the milk fat and/or the tissuefat of a ruminant which comprises administering to said ruminant thetrans-11 isomer of octadecenoic acid in an amount and for a period oftime effective to increase the CLA level to above that normally presentin the milk fat and/or tissue fat of a ruminant, optionally mixed withother feed, separately or together with other fatty acids.
 6. A methodaccording to claim 5, wherein the ruminant is a lactating ruminant.
 7. Amethod according to claim 6, wherein the trans-11 isomer of octadecenoicacid is protected from reduction in the rumen.
 8. A method according toclaim 5, wherein the other fatty acids are saturated fatty acids.
 9. Ananimal feed composition for increasing the concentration of the cis-9,trans-11 isomer of octadecadienoic acid (CLA) in the milk fat and/or thetissue fat of a ruminant, the composition comprising the trans-11 isomerof octadecenoic acid present in an amount effective to increase the CLAlevel to above that normally present in the milk fat and/or tissue fatof a ruminant.
 10. An animal feed according to claim 9, additionallycontaining other fatty acids.